Knowledge of cell immune function and tumor biology has shown that a competent immune system has a key role in cancer prevention and treatment.
Genetic and epigenetic events cause abnormal generation of a large number of new antigens (Ag), that are constantly produced by tumors as the latter develop and progress.
Immune system components may theoretically detect these Ags and trigger direct cell and tumor responses against the transformed cells, thereby inhibiting development thereof.
The anticancer immune response develops through various steps (see Fig. 1A), which require the presence of specific cytokines and/or chemokines, support cells such as antigen-presenting cells and cell effectors capable of tumor target-specific attack, by recognition of the Ag that activates the effector functions. Nevertheless, in spite of the immune response, immune cells cannot eliminate all the tumor cells, as the latter use a number of “escape” mechanisms (see FIG. 1B).
This generates somewhat an immune tolerance against tumor growth. Such events have been addressed by considering a number of therapeutic approaches, in which cell effectors are enhanced for more effective and persistent activity.
The most common therapeutic approaches include anticancer vaccination, adoptive cell immunotherapy and gene therapy.
In the first approach, cell effectors are caused to contact tumor-specific antigens and trigger response therefrom.
In adoptive immunotherapy, cell effectors are infused, after ex-vivo multiplication and purification thereof according to a selective anti-tumor activity.
Finally, gene therapy allows modification of cell effectors for more specific antigen recognition, followed by an enhanced and possibly more persistent anti-tumor response.
Such gene therapy has been tested for effectiveness against diseases such as melanoma, intestinal carcinoma, lymphoma, prostate cancer, kidney tumor, and even in acute and chronic lymphatic leukemia (FIG. 1C). These encouraging results suggested us to study a gene therapy approach using immune cells, that focused on tumor lines expressing a particular antigen, known as GD2, on their cell membrane.
Gene Therapy with Anti-GD2 Chemical Receptor-Expressing Immune Cells.
Disialoganglioside GD2 is a ubiquitous molecule of the plasma membrane of neuroectodermal cells, which has a role in cell growth and differentiation, as well as the maintenance of cell-matrix adhesion.
II GD2 has been extensively investigated for the last 30 years, as it was found to be an interesting surface epitope characterizing various types of tumors of ectodermal and mesodermal origin.
Particularly, it is over-expressed by most of neuroblastoma (NB) and melanoma cells, and is absent or expressed at low levels in normal tissues.
In pediatrics, neuroblastoma, hereinafter shortly referred to as NB, is one of the most intensively studied tumors, as it has a yearly incidence of 7-10 cases/million, and ranks third in incidence among pediatric malignancies after leukemia and central nervous system tumors.
While the survival rate for low- and medium-risk NB cases has been ascertained, prognosis for high-risk NB cases is still low (<15%). Although the various therapies against NB have increased initial response rates, it is still difficult to achieve long-lasting remission in patients with disseminated disease.
Recent immunotherapy trials against NB showed susceptibility thereof to the action of effector cells, and suggest these procedures as a new interesting therapeutic approach to support existing therapies, with the purpose of preventing recurrence and ensuring long-term disease control.
Based on these trials, a number of therapeutic strategies for enhancing cell effectors have been implemented for NB treatment.
The first immunotherapy approaches consisted in infusing patients with tumor-specific anti-GD2 monoclonal antibodies (MoAb in short), immune-modulating cytokines such as IL-2 and ex-vivo activated immune cells, as described for adoptive immunotherapy.
Concerning MoAbs, once the specific tumor antigen has been recognized, they can act at multiple levels, namely on the one hand by triggering a block by direct action, if the antigen is a functional receptor and on the other with an indirect action, through an Ab-dependent cytotoxicity process (ADCC), involving activation of cell effectors.
Furthermore, MoAbs have been conjugated with radioactive molecules, chemotherapeutic drugs or toxins, thereby acting as carriers for highly specific delivery of the active compound to the tumor site.
Also concerning NB, various clinical trials have been undertaken with anti-GD2 MoAbs (see Fig. 1D) to trigger an immune response, although the first results that have obtained have shown poor success in NB recurrence treatment as well as high toxicity.
In order to obviate these limits, adoptive cell immunotherapy has been undertaken, using disialoganglioside GD2 as a target antigen.
Immune cells have been modified for recognition and elimination of GD2-positive tumors, thereby providing new therapeutic hopes not only to children affected by this tumor, but also to patients with tumors associated with high GD2-antigen expression, such as: small cell lung carcinoma, melanoma and other tumors.
In 1 998, a New York-based team led by Sadelain M, noted that most tumor cells use an “escape” mechanism that consists in reduced presentation of molecules involved in co-stimulation to activation of the immune system, such as B7-1 and B7-2.
These molecules are required by the effector cells both for completing tumor cell recognition and for maintaining the established proliferative stimulus. Based on this assumption, a decision was made of modifying the T cells by providing them with a CR that is capable of recognizing GD2-positive cells, and consists of the scFv of a GD2-specific MoAb (3G6) associated with the co-stimulatory molecule CD28, which is required for effective activation of the anti-tumor action of lymphocytes.
The same chimeric receptor was produced in truncated form, i.e. lacking the intra-cytoplasmic portion, and was used as a control to validate the role of the CD28 molecule in the persistence of lymphocyte activation stimulus.
The first in vitro results showed that the CR 3G6-CD28 was not only able to allow specific recognition of GD2-positive cells by T lymphocytes, but could also protect them from death induced by tumor microenvironment stimuli, thereby allowing selective expansion of modified lymphocytes, unlike the ECs expressing the truncated-form CR.
Although this study provided one of the first evidences of the important role of co-stimulatory molecules in ensuring proliferation and long-term survival of immune cells, it did not deal with the ability of modified lymphocytes to act against tumor cells, and did not assess their effectiveness in terms of anti-tumor immune action.
The team of Brenner M. K. and Rossig C, from the Baylor College of Medicine, Huston, conducted a study in 2001, in which T lymphocytes were genetically modified and used in NB treatment.
Here, CRs were generated whose intracytoplasmic domain had only the lymphocyte signaling molecule CD3ζ (with no co-stimulatory signal), fused with an extracytoplasmic domain composed of the scFv of the anti-GD2 monoclonal antibody (sc14.G2a).
The T lynmphocytes obtained after transduction were able to stably express the CR with less than 30% efficiency, but this assessment, although not extended to all lymphocytes, ensured selective recognition and activation of such modified lymphocytes against GD2-positive NB cells.
In spite of this, their function was found to decrease with time, when no appropriate co-stimulatory signal was provided, because antigen stimulation by the CR was not found to be able, alone, to adequately support cell proliferation.
These results support the feasibility of human T-lymphocytes redirection for treatment of an associated GD2 tumor, but indicate that the success of CR-mediated adoptive immunotherapy requires additional strategies to support maintenance of EC activation.
More recent studies have shown that co-stimulatory molecules are required by immune cells not only to complete activation of the cytolytic action once the target (primary and secondary signals) is recognized, but to ensure such action with time.
These observations suggest that any gene modification of ECs presenting a CR in combination with a co-stimulatory signal may be a key to obtain specific and long-lasting immune responses.
These assumptions were used by Brenner M and his colleagues in 2008 to prove that cytotoxic T lymphocytes CD8+ (CTL) directed against tumor-associated non-viral antigens do not survive for a long time in the tumor microenvironment and show a limited in-vivo antitumor activity, whereupon the researchers decided to utilize their antiviral reactivity to make up for this limit.
Therefore, Epstein-Barr (EBV)-specific CTLs were genetically modified for expression of a CR directed against GD2 and hence these genetically modified lymphocytes receive the co-stimulatory signal from their native receptors activated by the EBV virus, while the specific antitumor activity is mediated by the CR.
This study conducted on NB patients showed that EBV-specific CTLs expressing the anti-GD2 CR survive for a longer time as compared both with the same CR-modified lymphocytes with no EBV specificity, and with CTLs having an anti-GD2 CR, that has the CD3ζ chain as the only intra-cytoplasmic stimulatory signal.
The infusion of EBV-specific CTLs modified with the anti-GD2 CR was found to be safe and was associated with tumor regression in one half of the tested individuals, which suggests it as an interesting adoptive cell immunotherapy approach, although in this case co-stimulation is not supported by an inherent property of the CR but by prior sensitization of ECs by the EB virus, which limits the field of action to individuals that had specific contact with such virus. Furthermore, individuals formerly exposed to the virus might have the virus in their T cells, as a condition that predisposes per se to development of lymphoid neoplasia.
Therefore, various co-stimulatory molecules have been studied and investigated through the years, for association to the CR and enhancement of its effectiveness. A remarkable molecule of this type is the 4-1 BB molecule, also known as CD137, and member of the TNF receptor family, which is essential for the antitumor activity of T cells.
In 2009, Campana D. et al. issued a study in which T lymphocytes were modified with an anti-CD19 CR (an important marker for acute lymphatic leukemia), which presented the 4-1 BB co-stimulatory molecule signaling domain at intra-cytoplasmic level.
The T lymphocytes genetically modified to express this CR have shown a potent and specific cytolytic activity against CD19-positive leukemia cells and such effectiveness was found to be better than that of lymphocytes modified with the same CR, lacking the 4-1 BB portion. Based on this and other previous studies, Campana D. et al. disclosed in patent application publication US2005/0113564 concerning “Chimeric receptors with 4-1 BB stimulatory signaling domain”, the possibility of genetically modifying ECs by providing them with specific anti-CD-19 CRs having a highly efficient co-stimulatory molecule, thereby optimizing the activation and maintenance of the antitumor cytolytic stimulus with time.
All these studies provide the knowledge that treatments with large numbers of genetically modified effector cells may be used in clinical practice, due to their low hazard level, although successful use of this therapy may be hindered by low long-term persistence of these cells, as well as by an advanced state of the tumor disease.
Such data suggest the need of improving the immunologic action after transplantation by modifying the receptor structure to optimize its ability of both first activating the antitumor cytolytic process; and allowing a more effective long-time persistence of the effector cells at the tumor site, for real, long-term remission of the disease.